The combination of the macrocyclic hosts p‐sulfonatocalix[4]arene and cucurbit[7]uril with the fluorescent dyes lucigenin and berberine affords two label‐free enzyme assays for the detection of kinase and phosphatase activity by fluorescence monitoring. In contrast to established assays, no substrate labeling is required. Since phosphorylation is one of the most important regulatory mechanisms in biological signal transduction, the assays should be useful for identification of inhibitors and activators in high‐throughput screening (HTS) format for drug discovery.